Toxoplasma gondii is an obligate intracellular parasite that causes severe disease in immunocompromised individuals and congenitally infected neonates. Toxoplasma also serves as a model system for the study of related apicomplexan parasites including Plasmodium falciparum, the causative agent of malaria. Intracellular survival of these organisms is critically dependent on the ability of the parasite to actively invade their host cell, establish a replication-permissive vacuole, and avoid host cell defenses. Apicomplexan parasites share a unique mechanism for invasion that involves the formation of a tight junction between the invading parasite and the host cell called the moving junction (MJ). The MJ is believed to form a stable anchor for the parasite to invade the host cell and also serve as a molecular sieve that modifies the nascent vacuole to render it non-fusogenic with the host endocytic pathway. The Toxoplasma MJ consists of micronemal AMA1 on the parasite's surface connected to a macromolecular complex of rhoptry neck proteins (RONs 2/4/5/8) that are injected into the host cell. While RON2 spans the host membrane and establishes the link to AMA1, the remaining members of the complex are surprisingly on the cytoplasmic face of the host membrane and how they function in invasion is largely unknown. We have disrupted the coccidial-specific component RON8 and shown that while not essential, this protein plays an important role in parasite invasion in vitro and in virulence in vivo. As the remaining members are conserved in the Apicomplexa and believed to be essential, this indicates that apicomplexans contain a conserved core complex that is required for invasion, which is enhanced in the coccidia via RON8. This is supported by our recent development of a conditional knockout for RON5, which shows that this protein plays a critical role in assembly of the complex and that the MJ complex is indeed essential for invasion. Our objectives in this first renewal application are to conduct an in depth functional analysis of the MJ complex and determine its architecture. Specifically, we will first focus on RON8 to determine how this component enhances invasion and links the complex to the host cell. We will then exploit the conditional knockout of RON5 to study its role in the organization and function of the MJ complex. Lastly, we will explore the architecture of the complex by determining its stoichiometry and identifying key interactions of its component proteins. These studies will open completely new insight into the mechanism by which apicomplexan parasites use this novel invasion machine to infect their mammalian hosts and cause disease.